The human genome is composed of > 50 % transposable elements, which are an important source of human genetic variation. 3% of the human genome consists of DNA-based (cut-and-paste) transposons and 43 of the 47 genes related to transposable elements are derived from these mobile elements. Most of these genes remain uncharacterized with the exception of the lymphoid-specific RAG1/RAG2 V(D) recombinase and the SETMAR protein, a fusion between a mariner transposase and a histone methylase SET domain. One member of this collection of transposon-related genes is called THAP9, which is homologous along its entire length to the Drosophila P element transposase protein. THAP9 and eleven other human genes contain a THAP domain, a newly recognized C2CH zinc-coordinating site-specific DNA binding domain. There are > 300 THAP domain-containing proteins in the genomes of eukaryotes, but only THAP9 is homologous beyond the THAP domain. Comparative genomic analyses indicate that the THAP9 gene is present in 21 vertebrate genomes surveyed, but is absent from rodents and that there are P element-like transposons with terminal inverted repeats in the zebrafish genome. These observations suggest that the THAP9 genes were derived from ancient transposons, much like the scenario for evolution of the V(D)J recombination- RAG1/RAG2 recombinase system. We wish to characterize the human THAP9 gene, which our preliminary data indicate is an active DNA transposase. The long-term goal of this research is to understand how genes related to transposable elements function in the human genome. Because transposable elements cause mutations, genome rearrangements, lead to genetic variation and alter gene expression, they are important components of genome evolution and gene expression programs. The experiments outlined in this proposal will characterize the functional activities of the THAP9 protein, and will potentially identify a new family of human transposable elements. The central hypothesis of this proposal is that the human THAP9 gene encodes an active DNA transposase. We will test this hypothesis in several ways and attempt to find active transposable elements in the human genome that use the THAP9 protein as a transposase. The rationale behind this proposal is to discover an active DNA-based transposon system in humans. In order to address the functional role THAP9 plays in human cells, we will: 1) Investigate and characterize the activities of human THAP9 in cells; 2) Map human THAP9 genomic DNA binding sites using chromatin immunoprecipitation-HTP sequencing (ChIP-seq); 3) Proteomic and biochemical analysis of human THAP9 protein.